Article Information

• PDF (1013 kb)

PubMed

• Articles by J.M. Vanderkooi
• Articles by G.V. Woodrow

Related Articles

• Solution Properties of Tetramethylrhodamine-Modified G-Actin...

  Solution Properties of Tetramethylrhodamine-Modified G-Actin Biophysical Journal, Volume 85, Issue 4, 1 October 2003, Pages 2466-2475 Dmitry S. Kudryashov and Emil Reisler Abstract In the recently solved structure of TMR-modified ADP-G-actin, the nucleotide cleft is in a closed state conformation, and the D-loop contains an -helix (L. R. Otterbein, P. Graceffa, and R. Dominguez, 2001, , 293:708–711). Subsequently, questions were raised regarding the possible role of the TMR label on Cys in determining these aspects of G-actin structure. We show here that the susceptibility of D-loop on G-actin to subtilisin cleavage, and ATP/ADP-dependent changes in this cleavage, are not affected by TMR-labeling of actin. The TMR modification inhibits nucleotide exchange, but has no effect on DNase I binding and the fast phase of tryptic digestion of actin. These results show an absence of allosteric effects of TMR on subdomain 2, while confirming ATP/ADP-dependent changes in D-loop structure. In conjunction with similar results obtained on actin-gelsolin segment 1 complex, this works reveals the limitations of solution methods in probing the putative open and closed nucleotide cleft states of G-actin. Abstract | Full Text | PDF (278 kb)

• The structure of bovine glutamate dehydrogenase provides ins...

  The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery Structure, Volume 7, Issue 7, 15 July 1999, Pages 769-782 Peter E Peterson and Thomas J Smith Summary We propose that the antenna serves as an intersubunit communication conduit during negative cooperativity and allosteric regulation. GTP and NADH inhibit GDH by keeping the catalytic cleft in a closed conformation. In contrast, ADP probably binds to the back of the NAD-binding domain and activates the enzyme by keeping the catalytic cleft open. Extensive contacts between antennae within the crystal lattice may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix. Summary | Full Text | PDF (312 kb)

• Reversible Inactivation of Myosin Subfragment 1 Activity by ...

  Reversible Inactivation of Myosin Subfragment 1 Activity by Mechanical Immobilization Biophysical Journal, Volume 74, Issue 3, 1 March 1998, Pages 1465-1472 Stefan Highsmith, Kelly Duignan, Kathy Franks-Skiba, Katherine Polosukhina and Roger Cooke Abstract The Mg-ATPase activity of skeletal muscle myosin subfragment 1 (S1) is reversibly eliminated when it is aggregated by the force of osmotic pressure dehydration using polyethylene glycol (PEG). Several experiments indicate nucleotides bind aggregated S1, but the effects of binding are attenuated. Compared with S1 in solution, ϵADP binds aggregated S1 with reduced affinity, and the bound ϵADP fluorescence intensity is more effectively quenched by acrylamide. When ATP binds aggregated S1, the tryptophan intensity increases to only 50% of the solution level. Chemical cross-linking of cys-707 to cys-697 by -phenylenedimaleimide is less efficient for aggregated S1·MgADP. The data are consistent with aggregated S1 being able to bind nucleotide but not being able to complete the usual conformation change(s) in response to binding. If S1 is kept from aggregating by increasing the ionic strength at the same osmotic pressure, its Mg-ATPase activity and ATP-induced tryptophan fluorescence intensity increase are normal. The combined data are consistent with an ATP hydrolysis mechanism in which S1 segmental motion is coupled to its enzymatic activity. In this model, segmental motion is mechanically constrained by aggregation; the constrained S1 can bind ATP, but it cannot complete the hydrolysis mechanism. Abstract | Full Text | PDF (175 kb)

• …more

Abstract

The fluorescence parameters of ethenoadenosine derivatives are influenced by metal cations and pH, as summarized here. The pH profile of ethenoadenosine determined by fluorescence intensity gives a normal titration curve and is not affected by ionic strength. In contrast, the pH titration curves of etheno-ATP, etheno ADP, and etheno AMP depend upon ionic strength. At high ionic strength normal curves are obtained, whereas at low ionic strength anomalies are obtained; this suggests that the phosphates can interact with the ring, possibly by hydrogen binding to the ring nitrogens. The room temperature fluorescence of ethenoadenosine occurs from the base form, although excitation of either the acid or base forms can contribute to the emission. This result can be explained if the excited state pK is lower than the ground state pK, and if deprotonation occurs within the time scale of the excited state. At low pH values the fluorescence lifetime of the base form is dependent upon the buffer concentration, indicating that the reverse reaction, protonation, occurs. The affinity constants for the binding of metals to the ethenoadenosine phosphates resemble those for the corresponding adenosine phosphates. Ni(II) and Co(II) are more effective than Mn(II) in quenching the fluorescence of ethenoadenosine phosphates; this result is predicted by Förster's theory for energy transfer based upon the overlap between donor emission spectrum and acceptor absorption spectrum. The diamagnetic ions Mg(II), Ca(II), and Zn(II) do not appear to affect the fluorescence of the ethenoadenosine phosphates directly, but rather to affect the conformation of the molecule, thereby affecting the quantum yield.